Maternal metabolic diseases are known to alter the intrauterine environment, with potential for unfavorable pregnancy outcomes, such as birth defects. Among the most severe defects are neural tube defects, which occur more frequently in pregnancies complicated by maternal obesity or diabetes. We here propose that exposure to an adverse intrauterine environment affects embryonic development through epigenetic mechanisms. The overarching hypothesis for this proposal is that maternal diabetes induces changes in histone acetylation that mediate altered transcriptional responses after exposure, thereby increasing the risk for neural tube defects. We will test this hypothesis by focusing on H3K9 and H3K27 acetylation, two hallmarks of active gene transcription. We propose to investigate the role of these chromatin modifications in the development of mesoderm, which we have shown to be disrupted during neural tube closure in two separate mouse models of diabetic pregnancy. Our Specific Aims are: 1) Determine the contribution of changes in histone acetylation to transcriptional responses to maternal hyperglycemia; 2) Define the role of exposure-induced epigenetic changes in mesoderm development in an embryonic stem cell differentiation model; 3) Investigate how epigenetic editing of individual chromatin marks at specific genomic loci affects transcription and mesoderm differentiation from embryonic stem cells. This will be combined with optogenetic approaches to experimentally modify neural tube defect risk in mice undergoing epigenetic editing in vivo. The direct manipulation of specific chromatin modifications in cells and mice is novel in concept, and several technologies we propose to use are highly innovative. The long-term goal of this research is to uncover the molecular mechanisms through which adverse exposures during pregnancy cause birth defects, and increase the potential for adverse health outcomes later in life. Identifying the targets and pathways involved in the response to harmful intrauterine conditions, such as those in diabetic pregnancies, has high significance for the prevention and treatment of unfavorable pregnancy outcomes, such as neural tube defects.